AAT Bioquest

Amplite® Fluorimetric Peroxidase (HRP) Assay Kit *Near Infrared Fluorescence*

HRP dose response was measured with Amplite® Fluorimetric Peroxidase Assay Kit in a solid black 384-well plate using a Gemini fluorescence microplate reader (Molecular Devices).
HRP dose response was measured with Amplite® Fluorimetric Peroxidase Assay Kit in a solid black 384-well plate using a Gemini fluorescence microplate reader (Molecular Devices).
HRP dose response was measured with Amplite® Fluorimetric Peroxidase Assay Kit in a solid black 384-well plate using a Gemini fluorescence microplate reader (Molecular Devices).
Ordering information
Catalog Number
Unit Size
Add to cart
Additional ordering information
InternationalSee distributors
Bulk requestInquire
Custom sizeInquire
ShippingStandard overnight for United States, inquire for international
Request quotation
Spectral properties
Excitation (nm)648
Emission (nm)668
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22


Excitation (nm)
Emission (nm)
Peroxidase is a small molecule (MW ~40 KD) that can usually be conjugated to an antibody in a 4:1 ratio. Due to its small size, it rarely causes steric hindrance problem with antibody/antigen complex formation. Peroxidase is inexpensive compared to other labeling enzymes. The major disadvantage associated with peroxidase is their low tolerance to many preservatives such as sodium azide that inactivates peroxidase activity even at low concentration. HRP conjugates are extensively used as secondary detection reagents in ELISAs, immuno-histochemical techniques and Northern, Southern and Western blot analyses. We offer this quick (10 min) HRP assay in a one-step, homogeneous, no wash assay system. This kit uses Amplite® IR, our near infrared flurogenic HRP substrate. Amplite® IR generates a substance that has maximum absorption of 647 nm with maximum emission at 670 nm. This near infrared absorption and fluorescence minimize the assay background that is often caused by the autoabsorption and/or autofluorescence of biological samples that rarely absorb light beyond 600 nm. The kit can be used for ELISAs, characterizing kinetics of enzyme reaction and high throughput screening of oxidase inhibitors, etc. The kit provides an optimized 'mix and read' assay protocol that is compatible with HTS liquid handling instruments.


Absorbance microplate reader

Absorbance647 ± 5 nm
Recommended plateClear bottom

Fluorescence microplate reader

Emission680 nm
Cutoff665 nm
Recommended plateSolid black


Example protocol


Protocol Summary
  1. Prepare HRP standards and/or test samples (50 µL)
  2. Add Peroxidase working solution (50 µL)
  3. Incubate at room temperature for 30 - 60 minutes
  4. Monitor fluorescence intensity at Ex/Em = 640/680 nm (Cutoff = 665 nm) 
Important      Thaw all the kit components at room temperature before starting the experiment. The component A is unstable in the presence of thiols such as DTT and β-mercaptoethanol. The presence of thiols at concentration higher than 10 µM would significantly decrease the assay dynamic range. NADH and glutathione (reduced form: GSH) may interfere with the assay.


Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. Amplite™ IR Peroxidase Substrate stock solution (100X)
Add 250 µL of DMSO (Component E) into the vial of Amplite™ IR Peroxidase Substrate (Component A) to make 100X Amplite™ IR Peroxidase Substrate stock solution .

2. HRP standard solution (20 U/mL)
Add 1 mL of Assay Buffer (Component C) into the vial of Horseradish Peroxidase (Component D) to make 20 U/mL HRP standard solution.

3. H2O2 stock solution (20 mM)
Add 22.7 µL of 3% H2O2 (0.88 M, Component B) into 977 µL of Assay Buffer (Component C) to make 20 mM H2O2 stock solution. Note: The diluted H2O2 solution is not stable. The unused portion should be discarded.


For convenience, use the Serial Dilution Planner:

HRP standard
Add 15 µL of 20 U/mL HRP standard solution into 985 µL of Assay Buffer (Component C) to get 300 mU/mL HRP standard solution (SD7). Take 300 mU/mL HRP standard solution (SD7) and perform 1:3 serial dilutions to get serially diluted HRP standards (SD6 - SD1) with Assay Buffer (Component C).


Add 50 μL of 100X Amplite™ IR Peroxidase Substrate stock solution and 50 μL of 20 mM H2O2 stock solution into 4.9 mL of Assay Buffer (Component C) to make Peroxidase working solution. Keep from light.


Table 1. Layout of HRP standards and test samples in a solid black 96-well microplate. SD= HRP Standards (SD1 - SD7, 0.41 to 300 mU/mL), BL=Blank Control, TS=Test Samples.
Table 2. Reagent composition for each well.
SD1 - SD750 µLSerial Dilutions (0.41 to 300 mU/mL)
BL50 µLAssay Buffer (Component C)
TS50 µLtest sample
  1. Prepare HRP standards (SD), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.
  2. Add 50 µL of Peroxidase working solution to each well of HRP standard, blank control, and test samples to make the total Peroxidase assay volume of 100 µL/well. For a 384-well plate, add 25 µL of Peroxidase working solution into each well instead, for a total volume of 50 µL/well.
  3. Incubate the reaction at room temperature for 30 to 60 minutes, protected from light.
  4. Monitor the fluorescence increase with a fluorescence plate reader at Excitation = 600 - 650 nm, Emission = 650 - 690 nm (optimal Ex/Em = 640/680 nm, Cutoff = 665 nm). Note: The contents of the plate can also be transferred to a white clear bottom plate and read by an absorbance microplate reader at the wavelength of 647 ± 5 nm. The absorption detection has lower sensitivity compared to fluorescence reading. 


Open in Advanced Spectrum Viewer

Spectral properties

Excitation (nm)648
Emission (nm)668

Product Family



View all 1 citations: Citation Explorer
Identification of acetylcholinesterase inhibitors using homogenous cell-based assays in quantitative high-throughput screening platforms
Authors: Li, Shuaizhang and Huang, Ruili and Solomon, Samuel and Liu, Yitong and Zhao, Bin and Santillo, Michael F and Xia, Menghang
Journal: Biotechnology Journal (2017): 1600715


View all 49 references: Citation Explorer
Horseradish peroxidase-driven fluorescent labeling of nanotubes with quantum dots
Authors: Didenko VV, Baskin DS.
Journal: Biotechniques (2006): 295
Enzymatic oxidation of dipyridamole in homogeneous and micellar solutions in the horseradish peroxidase-hydrogen peroxide system
Authors: Almeida LE, Imasato H, Tabak M.
Journal: Biochim Biophys Acta (2006): 216
Recent advances in catalytic peroxidase histochemistry
Authors: Krieg R, Halbhuber KJ.
Journal: Cell Mol Biol (Noisy-le-grand) (2003): 547
Vesicular transport route of horseradish C1a peroxidase is regulated by N- and C-terminal propeptides in tobacco cells
Authors: Matsui T, Nakayama H, Yoshida K, Shinmyo A.
Journal: Appl Microbiol Biotechnol (2003): 517
Synthesis and purification of horseradish peroxidase-labeled oligonucleotides for tyramide-based fluorescence in situ hybridization
Authors: van Gijlswijk RP, van de Corput MP, Bezrookove V, Wiegant J, Tanke HJ, Raap AK.
Journal: Histochem Cell Biol (2000): 175
Preparation, morphological characterization, and activity of thin films of horseradish peroxidase
Authors: Vianello F, Zennaro L, Di Paolo ML, Rigo A, Malacarne C, Scarpa M.
Journal: Biotechnol Bioeng (2000): 488
Development of a novel enzyme/prodrug combination for gene therapy of cancer: horseradish peroxidase/indole-3-acetic acid
Authors: Greco O, Folkes LK, Wardman P, Tozer GM, Dachs GU.
Journal: Cancer Gene Ther (2000): 1414
In situ identification of cyanobacteria with horseradish peroxidase-labeled, rRNA-targeted oligonucleotide probes
Authors: Schonhuber W, Zarda B, Eix S, Rippka R, Herdman M, Ludwig W, Amann R.
Journal: Appl Environ Microbiol (1999): 1259
Evidence for free radical formation during the oxidation of 2'-7'-dichlorofluorescin to the fluorescent dye 2'-7'-dichlorofluorescein by horseradish peroxidase: possible implications for oxidative stress measurements
Authors: Rota C, Chignell CF, Mason RP.
Journal: Free Radic Biol Med (1999): 873
Relative number of cells projecting from contralateral and ipsilateral nucleus isthmi to loci in the optic tectum is dependent on visuotopic location: horseradish peroxidase study in the leopard frog
Authors: Dudkin EA, Gruberg ER.
Journal: J Comp Neurol (1999): 212